Periodontal ligament fibroblasts (PDLFs) are important cells, which are involved in maintaining tooth integrity. Diabetes has been found to be associated with periodontal disease in a bidirectional manner. The aim of the present study was to investigate the stemness properties of human PDLFs (HPDLFs) in high glucose conditions. HPDLFs were analyzed for their osteogenic differentiation capacity by inducing the cells with osteogenic medium in various glucose concentrations. The gene expression was then examined using reverse transcription‑quantitative polymerase chain reaction analysis, and examinations of alkaline phosphatase activity and nodule formation were performed. The results of the gene expression analysis revealed that high glucose media induced the expression of NANOG, octamer-binding transcription factor 4, (sex determining region Y)‑box 2, cluster of differentiation 166 (CD166), PERIOSTIN and β‑CATENIN following culture of the cells for 3 days. Alkaline phosphatase activity increased following 14 days in the high glucose condition. In addition, higher numbers of calcified nodules were formed on day 28 in the group cultured with high glucose. The results showed that high glucose induced bone formation by elevating the expression of stem cell markers, particularly CD166, and this induction may be regulated through β-CATENIN.

Porphyromonas gingivalis (P. gingivalis) and nicotine have been implicated as a major pathogen in the development and progression of periodontitis. One of the possible mechanism is via the oxidative stress of human periodontal ligament fibroblasts (PDLF) which lead to the damage of cell viability and function. This study aimed to investigate oxidative stress (OS) levels in the cultured media of human PDLF under the induction of P. gingivalis lysate and nicotine. Primary PDLF was cultured in growth media under P. gingivalis or/and nicotine treatment in different concentrations for 2 and 24 h. Following incubation, oxidative stress molecules malondialdehyde (MDA) and oxidized guanine species (Ox-GS) from the cell cultured supernatant were determined by spectrophotometric assay and ELISA, respectively. DCFDA and superoxide assays were performed to verify the production of ROS and intracellular superoxide radical under various stimuli. As a result, at both 2 and 24 h, Ox-GS and MDA levels in the medium of cells treated with different concentrations of P. gingivalis lysate and nicotine, either separately or in combination, were significantly different from the negative controls in a dose- and time-dependent manner. Interestingly, except MDA levels in P. gingivalis lysate at 20 µg/ml, MDA levels in all other tested conditions were found as same as one in the positive controls after 24 h. ROS and superoxide production were enhanced under P. gingivalis and/or nicotine stimulation. Therefore, OS biomarkers were generated by PDLF upon treatment with periodontal pathogens and nicotine which could elucidate a potential local mechanism of periodontal disease etiology via superoxide mediation.